Abstract | OBJECTIVE: METHODS: Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays. RESULTS: Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus. CONCLUSION: This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.
|
Authors | Hua Zhang, Juan Feng, Hongsheng Chen, Jiada Li, Hunjin Luo, Yong Feng |
Journal | Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics
(Zhonghua Yi Xue Yi Chuan Xue Za Zhi)
Vol. 32
Issue 6
Pg. 805-9
(Dec 2015)
ISSN: 1003-9406 [Print] China |
PMID | 26663053
(Publication Type: English Abstract, Journal Article, Research Support, Non-U.S. Gov't)
|
Chemical References |
- Microphthalmia-Associated Transcription Factor
- Nuclear Localization Signals
- Green Fluorescent Proteins
- Luciferases
- Monophenol Monooxygenase
|
Topics |
- Amino Acid Sequence
- Animals
- Cell Line, Tumor
- Genetic Predisposition to Disease
(genetics)
- Green Fluorescent Proteins
(genetics, metabolism)
- Humans
- Luciferases
(genetics, metabolism)
- Mice
- Microphthalmia-Associated Transcription Factor
(genetics, metabolism)
- Microscopy, Confocal
- Monophenol Monooxygenase
(genetics, metabolism)
- Mutation
- NIH 3T3 Cells
- Nuclear Localization Signals
(genetics)
- Transcriptional Activation
- Transfection
- Waardenburg Syndrome
(diagnosis, genetics, metabolism)
|